scholarly journals Gezielte Zellsortierung in der Einzelzellgenomik

BIOspektrum ◽  
2021 ◽  
Vol 27 (3) ◽  
pp. 274-276
Author(s):  
Morgan S. Sobol ◽  
Anne-Kristin Kaster
Keyword(s):  
Dark Matter ◽  
Single Cell ◽  
Cell Sorting ◽  
Genetic Information ◽  
Fluorescent Labeling ◽  
Single Cell Genomics ◽  
Rare Biosphere ◽  
Prokaryotic Genomes ◽  
Selection Of

AbstractSingle cell genomics (SCG) can provide reliable context for assembled genome fragments on the level of individual prokaryotic genomes and has rapidly emerged as an essential complement to cultivation-based and metagenomics research approaches. Targeted cell sorting approaches, which enable the selection of specific taxa by fluorescent labeling, compatible with subsequent single cell genomics offers an opportunity to access genetic information from rare biosphere members which would have otherwise stayed hidden as microbial dark matter.

scholarly journals Printing Microbial Dark Matter: Using Single Cell Dispensing and Genomics to Investigate the Patescibacteria/Candidate Phyla Radiation

2021 ◽  
Vol 12 ◽  
Author(s):  
Sandra Wiegand ◽  
Hang T. Dam ◽  
Julian Riba ◽  
John Vollmers ◽  
Anne-Kristin Kaster
Keyword(s):  
Dark Matter ◽  
Single Cell ◽  
Cell Sorting ◽  
Treatment Plant ◽  
Rrna Gene ◽  
Label Free ◽  
Single Cell Genomics ◽  
Sorting Technique ◽  
Unseen Species ◽  
Candidate Phyla Radiation

As of today, the majority of environmental microorganisms remain uncultured. They are therefore referred to as “microbial dark matter.” In the recent past, cultivation-independent methods like single-cell genomics (SCG) enabled the discovery of many previously unknown microorganisms, among them the Patescibacteria/Candidate Phyla Radiation (CPR). This approach was shown to be complementary to metagenomics, however, the development of additional and refined sorting techniques beyond the most commonly used fluorescence-activated cell sorting (FACS) is still desirable to enable additional downstream applications. Adding image information on the number and morphology of sorted cells would be beneficial, as would be minimizing cell stress caused by sorting conditions such as staining or pressure. Recently, a novel cell sorting technique has been developed, a microfluidic single-cell dispenser, which assesses the number and morphology of the cell in each droplet by automated light microscopic processing. Here, we report for the first time the successful application of the newly developed single-cell dispensing system for label-free isolation of individual bacteria from a complex sample retrieved from a wastewater treatment plant, demonstrating the potential of this technique for single cell genomics and other alternative downstream applications. Genome recovery success rated above 80% with this technique—out of 880 sorted cells 717 were successfully amplified. For 50.1% of these, analysis of the 16S rRNA gene was feasible and led to the sequencing of 50 sorted cells identified as Patescibacteria/CPR members. Subsequentially, 27 single amplified genomes (SAGs) of 15 novel and distinct Patescibacteria/CPR members, representing yet unseen species, genera and families could be captured and reconstructed. This phylogenetic distinctness of the recovered SAGs from available metagenome-assembled genomes (MAGs) is accompanied by the finding that these lineages—in whole or in part—have not been accessed by genome-resolved metagenomics of the same sample, thereby emphasizing the importance and opportunities of SCGs.

scholarly journals Targeted Cell Sorting Combined With Single Cell Genomics Captures Low Abundant Microbial Dark Matter With Higher Sensitivity Than Metagenomics

2020 ◽  
Vol 11 ◽  
Author(s):  
Hang T. Dam ◽  
John Vollmers ◽  
Morgan S. Sobol ◽  
Angela Cabezas ◽  
Anne-Kristin Kaster
Keyword(s):  
Dark Matter ◽  
Single Cell ◽  
Cell Sorting ◽  
Single Cell Genomics ◽  
Higher Sensitivity

Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies

2017 ◽  
Vol 89 (7) ◽  
pp. 4007-4012 ◽  
Author(s):  
Martin Dippong ◽  
Peter Carl ◽  
Christine Lenz ◽  
Jörg A. Schenk ◽  
Katrin Hoffmann ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Single Cell ◽  
Cell Sorting ◽  
Cell Selection ◽  
Fluorescence Activated Cell Sorting ◽  
Selection Of

scholarly journals Single-cell Genomics: Defining Microbiology's Dark Matter

BioTechniques ◽  
2012 ◽  
Vol 52 (5) ◽  
pp. 301-303 ◽  
Author(s):  
Jeffrey M. Perkel
Keyword(s):  
Dark Matter ◽  
Single Cell ◽  
Single Cell Genomics

scholarly journals Function-driven single-cell genomics uncovers cellulose-degrading bacteria from the rare biosphere

2019 ◽  
Vol 14 (3) ◽  
pp. 659-675 ◽  
Author(s):  
Devin F. R. Doud ◽  
Robert M. Bowers ◽  
Frederik Schulz ◽  
Markus De Raad ◽  
Kai Deng ◽  
...  
Keyword(s):  
Microbial Community ◽  
Single Cell ◽  
Cellulose Degradation ◽  
Geothermal Field ◽  
Rrna Gene ◽  
Cellulolytic Activity ◽  
Single Cell Genomics ◽  
Rare Biosphere ◽  
Degrading Bacteria

AbstractAssigning a functional role to a microorganism has historically relied on cultivation of isolates or detection of environmental genome-based biomarkers using a posteriori knowledge of function. However, the emerging field of function-driven single-cell genomics aims to expand this paradigm by identifying and capturing individual microbes based on their in situ functions or traits. To identify and characterize yet uncultivated microbial taxa involved in cellulose degradation, we developed and benchmarked a function-driven single-cell screen, which we applied to a microbial community inhabiting the Great Boiling Spring (GBS) Geothermal Field, northwest Nevada. Our approach involved recruiting microbes to fluorescently labeled cellulose particles, and then isolating single microbe-bound particles via fluorescence-activated cell sorting. The microbial community profiles prior to sorting were determined via bulk sample 16S rRNA gene amplicon sequencing. The flow-sorted cellulose-bound microbes were subjected to whole genome amplification and shotgun sequencing, followed by phylogenetic placement. Next, putative cellulase genes were identified, expressed and tested for activity against derivatives of cellulose and xylose. Alongside typical cellulose degraders, including members of the Actinobacteria, Bacteroidetes, and Chloroflexi, we found divergent cellulases encoded in the genome of a recently described candidate phylum from the rare biosphere, Goldbacteria, and validated their cellulase activity. As this genome represents a species-level organism with novel and phylogenetically distinct cellulolytic activity, we propose the name Candidatus ‘Cellulosimonas argentiregionis’. We expect that this function-driven single-cell approach can be extended to a broad range of substrates, linking microbial taxonomy directly to in situ function.

scholarly journals Complementary Metagenomic Approaches Improve Reconstruction of Microbial Diversity in a Forest Soil

mSystems ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
L. V. Alteio ◽  
F. Schulz ◽  
R. Seshadri ◽  
N. Varghese ◽  
W. Rodriguez-Reillo ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
Single Cell ◽  
Forest Soil ◽  
Carbon Metabolism ◽  
Cell Sorting ◽  
Phylogenetic Diversity ◽  
Metabolic Potential ◽  
Shotgun Metagenomics ◽  
Rare Biosphere ◽  
Depth Analysis

ABSTRACT Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this “uncultivated majority” remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes. Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages. IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism.

Impact of single-cell genomics and metagenomics on the emerging view of extremophile “microbial dark matter”

Extremophiles ◽  
2014 ◽  
Vol 18 (5) ◽  
pp. 865-875 ◽  
Author(s):  
Brian P. Hedlund ◽  
Jeremy A. Dodsworth ◽  
Senthil K. Murugapiran ◽  
Christian Rinke ◽  
Tanja Woyke
Keyword(s):  
Dark Matter ◽  
Single Cell ◽  
Single Cell Genomics

scholarly journals Single-cell genomics of marine plankton: Studying the single life of eukaryotic microbes

2014 ◽  
Vol 36 (1) ◽  
pp. 16-22 ◽  
Author(s):  
Debashish Bhattacharya ◽  
Rajat S. Roy ◽  
Dana C. Price ◽  
Alexander Schliep
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Cell Sorting ◽  
Single Cells ◽  
Biotic Interactions ◽  
Food Vacuole ◽  
Single Cell Genomics ◽  
True Diversity ◽  
High Throughput Dna Sequencing ◽  
Eukaryotic Microbes

The oceans are full of innumerable numbers of single cells living in microenvironments. Understanding who they are, what they eat and what infects them can inform us about the true diversity of plankton, their biotic interactions and how they may respond to a changing environment. Analysing to significant depth the genomes and ‘gut’ (i.e. the food vacuole and other contents) of individual wild-caught cells would have been thought impossible only a few years ago. However, the rapidly expanding field of single-cell genomics, powered by modern cell-sorting procedures, high-throughput DNA sequencing and bioinformatics methods holds the promise to revolutionize understanding of the biodiversity and ecology of eukaryotic microbes and their places in the tree of life.

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